The present invention relates to compounds, compositions and methods for the treatment of papilloma virus (PV) infection, particularly human papilloma virus (HPV). In particular, the present invention provides novel indane derivatives, pharmaceutical compositions containing such derivatives and methods for using these compounds in the treatment of papilloma virus infection. More particularly, the present invention provides compounds, compositions and methods for inhibiting papilloma virus DNA replication by interfering with the E1-E2-DNA complex during initiation of DNA replication.
Papillomaviruses are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been identified in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincottxe2x80x94Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into xe2x80x9clow riskxe2x80x9d and xe2x80x9chigh riskxe2x80x9d types. The low risk types include HPV type 6, type 11 and type 13 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States there are 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11. About 90% of SIL is also caused by low risk types 6 and 11. The other 10% of SIL is caused by high risk HPVs.
The high risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO2 laser, electrosurgery, or surgical excision. Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunotherapy is also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for HPV infection since recurrent warts are common with all current therapies (Beutner and Ferenczy, 1997, Amer. J. Med., 102(5A), 28-37).
The ineffectiveness of the current methods to treat HPV infections has demonstrated the need to identify new means to control or eliminate such infections. In recent years, efforts have been directed towards finding antiviral compounds, and especially compounds capable of interfering with viral replication at the onset of infection (Hughes, 1993, Nucleic Acids Res. 21:5817-5823).
The life cycle of PV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, the cellular DNA replication machinery is maintained as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation the viral genome copy number and viral gene expression in turn increase, with the eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, supra).
The coding strands for each of the papillomavirus contain approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs based on their location in the genome. E1 to E8 are expressed early in the viral replication cycle, and two late genes (L1 and L2) encode the major and minor capside proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 are expressed in connection with host cell proliferation. The L1 and L2 gene products are involved in virion structure. The function of the E3, E4 and E8 gene products is uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are both essential and sufficient for viral DNA replication in vitro (Kuo et al., 1994, J. Biol. Chem. 30:24058-24065). This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-sequences of all papillomaviruses (PV) regardless of the viral species and type (Kuo et al., 1994, supra).
Evidence emanating from studies of BPV-1 have shown that E1 possesses ATPase and helicase activities that are required in the initiation of viral DNA replication (Seo et al., 1993a, Proc. Natl. Acad. Sci. USA 90:702-706; Yang et al., 1993, Proc. Natl. Acad. Sci. 90:5086-5090; and MacPherson et al., 1994, 204:403-408).
The E2 protein is a transcriptional activator that binds to E1 protein and forms a complex that binds specifically to the ori sequence (Mohr et al., 1990, Science 250:1694-1699). It is believed that E2 enhances binding of E1 to the BPV origin of replication (Seo et al., 1993b, Proc. Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested that E2 stabilizes E1 binding to the ori (1995, J. Biol. Chem., 270(45):27283-27291).
To thwart this disease, a chemical entity that would interfere with viral DNA replication is therefore desirable. The present invention therefore provides such compounds, compositions or methods that inhibit papilloma viral replication. More particularly, the compounds and composition of the present invention interfere with the E1-E2-DNA complex during the viral replication cycle.
The present description refers to a number of documents, the content of which is herein incorporated by reference.
In a first aspect, the invention provides a compound of formula (I), or its enantiomers or diastereoisomers thereof: 
wherein:
A is a 5- or 6-membered homocyclic ring, or a 5- or 6-membered heterocyclic ring containing 1 or more heteroatoms selected from N, O and S;
X is H and W is OH; or X and W together form a carbonyl group or an epoxide;
R1 is H; or one or two substituents independently selected from the group consisting of: hydroxy; halo; lower alkyl; lower alkoxy; lower thioalkyl; haloalkyl (e.g. trifluoromethyl); or xe2x80x94C(O)R2 wherein R2 is lower alkyl, aryloxy or benzyloxy;
Y is phenyl optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R6 is lower alkyl, lower cycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ring being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is a heterocycle (Het) containing one or more heteroatom selected from N, O or S, said Het optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is ethylene-phenyl, said ethylene moiety being optionally mono- substituted with lower alkyl, wherein said phenyl ring is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said phenyl ring being optionally fused with a saturated or unsaturated 4- to 6-membered ring optionally containing a heteroatom selected from N, O and S;
or Y is ethylene-Het, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein Het is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S;
R3 is selected from the group consisting of: lower alkyl, lower cycloalkyl, lower alkylene, aryl or lower aralkyl, all of which optionally mono- or di-substituted with:
lower alkyl, lower cycloalkyl, haloalkyl, halo, CN, azido, lower alkoxy, (lower alkyl)acyl, C1-6 thioalkyl, C1-6 alkylsulfonyl, NHC(O)-lower alkyl, NHC(O)-aryl, NHC(O)xe2x80x94O-lower alkyl, NHC(O)O-aryl, aryl, aryloxy, hydroxy, nitro, amino, or Het, said Het optionally mono- or di-substituted with lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile, trifluoromethyl, C(O)R6 wherein R6 is as defined above;
said lower cycloalkyl, aryl, lower aralkyl or Het being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S; and
R4 is a carboxylic acid, a salt or an ester thereof;
and wherein wavy lines represent bonds of unspecified stereochemistry;
and with the provisos that:
(1) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 4-methylphenyl, then R3 cannot be benzyl, 3-fluorophenyl, or 4-nitrophenyl;
(2) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and R3 is cyclohexyl, then Y cannot be 4-iodophenyl or 4-methylphenyl;
(3) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 4-fluorophenyl, then R3 cannot be 4-ethyloxycarbonylphenyl;
(4) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 2-methylphenyl then R3 cannot be 4-nitrophenyl;
(5) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 2-methylphenyl, then R3 cannot be phenyl or 2-bromo-4-methylphenyl;
(6) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 4-chlorophenyl, then R3 cannot be 2-methoxyphenyl or 1,3-benzodioxolyl;
(7) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is 4-ethylphenyl, then R3 cannot be 3-fluorophenyl; and
(8) when A is benzene, R1 is hydrogen, X and W together form a carbonyl group and Y is phenyl, then R3 cannot be phenyl.
Alternatively, the first aspect of the invention provides compounds having the following formulae, selected from the group consisting of: 
wherein A, X, W, R1, Y, R3 and R4 are as defined above, with the provisos indicated above.
Compounds of the invention may also be represented by formula I in forms (2) and (3): 
wherein A, X, W, R1, Y and R3 are as defined above, with the provisos indicated above.
As will be recognized by persons skilled in the art, the compounds in forms (2) and (3) are readily converted to compounds of formula (I) in form (1). Without wishing to be bound by theory, it is believed that the compounds of formula (I) are in equilibrium between forms (1), (2) or (3) depending on the solvent and the pH in which they are dissolved. It has however been demonstrated that compounds of formula (I) are biologically active in form (1), and that the compounds in forms (2) and (3) will hydrolyze in conditions reproducing mammalian plasma (pH 7.4), to yield biologically active form (1).
In a second aspect, the invention provides a pharmaceutical composition comprising an anti-papillomavirus virally effective amount of a compound of formula (I) or a therapeutically acceptable salt or ester thereof, in admixture with a pharmaceutically acceptable carrier medium or auxiliary agent.
In a third aspect, the invention provides a method for treating a papillomavirus viral infection in a mammal by administering to the mammal an anti-papilloma virus virally effective amount of the compound of formula (I) or a therapeutically acceptable salt or ester thereof, or a composition as described above, (all without the provisos indicated above for formula (I).
In fourth aspect, the invention provides a method for inhibiting the replication of papillomavirus by exposing virally infected cells to an amount of the compounds of formula (I) inhibiting the papilloma virus E1-E2-DNA complex, or a therapeutically acceptable salt or ester thereof, or a composition as described above, (all without the provisos indicated above for formula (I).
In a fifth aspect, the invention provides a use of compounds of formula (I) (without the provisos indicated above for formula (I)) for the manufacture of a medicament for treating a papillomavirus viral infection,.
In an sixth aspect, the invention provides a method of preventing perinatal transmission of HPV from mother to baby, by administering a compound of formula (I) (without the provisos indicated above for formula (I)) to the mother prior to giving birth.
In a seventh aspect, the invention provides a use of compounds of formula (I) (without the provisos indicated above for formula (I)) for the manufacture of a medicament for preventing perinatal transmission of HPV from mother to baby prior to giving birth.
In an eighth aspect, the invention provides an intermediate compound of formula (vi): 
wherein Y and R3 are as defined above, or enantiomers or diastereoisomers thereof, with the provisos indicated above for formula (I).
In a ninth aspect, the invention provides an intermediate compound of formula (xx), said compound having trans/trans relative stereochemistry: 
wherein Y, R3, and R4 are as defined above, with the provisos indicated above for formula (I).
In an tenth aspect, the invention provides an intermediate compound of formula (xxvi): 
wherein R1, R3 and Y are as defined above,
or a salt or an ester thereof, or enantiomers and diastereoisomers thereof, without the provisos indicated above for formula (I).
In a eleventh aspect, the invention provides an intermediate compound of formula (xxxii): 
wherein R1, R3 and Y are as defined above,
or a salt or an ester thereof, or enantiomers and diastereoisomers thereof, without the provisos indicated above for formula (I).
In a twelfth aspect, the invention provides a process for producing compounds of formula Ixe2x80x2, 
wherein X, R1, W, Y, R3, and R4 are as defined above, with the provisos indicated above for formula (I), comprising:
a) hydrolyzing, in a mixture of aqueous base and a co-solvent, either intermediate compound vi or intermediate compound xx 
to produce compounds of formula Ixe2x80x2, wherein R3, R4, and Y are as defined above.
In a thirteenth aspect, the invention provides a process for producing compounds of formula Ixe2x80x3, 
wherein X and W together form a carbonyl group, R4 is a carboxylic acid or an ester, and R1, Y, and R3 are as defined above, without the provisos indicated above for formula (I), comprising:
a) hydrolyzing, in a mixture of aqueous base and a co-solvent, intermediate compound xxvi, 
so as to produce compounds of formula Ixe2x80x3, wherein R1, Y, and R3 are as defined above.
In a fourteenth aspect, the invention provides, a process for producing compounds of formula Ixe2x80x2xe2x80x3, 
wherein X and W together form a carbonyl group, R4 is a carboxylic acid or an ester, and R1, Y, and R3 are as defined above, without the provisos indicated above for formula (I), comprising:
a) hydrolyzing, in a mixture of aqueous base and a co-solvent, intermediate compound xxxii 
so as to produce compounds of formula Ixe2x80x2xe2x80x3, wherein R1, Y, and R3 are as defined above.
Definitions
As used herein, the following definitions apply unless otherwise noted:
The term xe2x80x9chaloxe2x80x9d as used herein means a halo radical selected from bromo, chloro, fluoro or iodo.
The term xe2x80x9clower alkylxe2x80x9d (or C1-6 alkyl) as used herein, either alone or in combination with another radical, means straight or branched-chain alkyl radicals containing up to six carbon atoms and includes methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl. The term xe2x80x9cC0-6 alkylxe2x80x9d preceding a radical means that this radical can optionally be linked through a C1-6 alkyl radical or the alkyl may be absent (C0).
The term xe2x80x9clower cycloalkylxe2x80x9d as used herein, either alone or in combination with another radical, means saturated cyclic hydrocarbon radicals containing from three to seven carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term xe2x80x9clower alkoxyxe2x80x9d as used herein means straight chain alkoxy radicals containing one to four carbon atoms and branched chain alkoxy radicals containing three to four carbon atoms and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter radical is known commonly as tert-butoxy.
The term xe2x80x9chaloalkylxe2x80x9d as used herein means alkyl radical containing one to six carbon atoms wherein one or more hydrogen atom is replaced by a halogen atom (e.g. trifluoromethyl).
The term xe2x80x9caminoxe2x80x9d as used herein means an amino radical of formula xe2x80x94NH2. The term xe2x80x9clower alkylaminoxe2x80x9d as used herein means alkylamino radicals containing one to six carbon atoms and includes methylamino, propylamino, (1-methylethyl)amino and (2-methylbutyl)amino. The term xe2x80x9cdi(lower alkyl)aminoxe2x80x9d means an amino radical having two lower alkyl substituents each of which contains one to six carbon atoms and includes dimethylamino, diethylamino, ethylmethylamino and the like.
The term xe2x80x9cacylxe2x80x9d as used herein, either alone or in combination with another radical, refers to groups xe2x80x94C(O)R, wherein R is lower alkyl or lower alkoxy.
The term xe2x80x9cC6 or C10 arylxe2x80x9d as used herein, either alone or in combination with another radical, means either an aromatic monocyclic system containing 6 carbon atoms or an aromatic cyclic system containing 10 carbon atoms. For example, aryl includes phenyl or naphthalene.
The term xe2x80x9cC7-16 aralkylxe2x80x9d as used herein, either alone or in combination with another radical, means an aryl as defined above linked through an alkyl group, wherein alkyl is as defined above containing from 1 to 6 carbon atoms. Aralkyl includes for example benzyl, and butylphenyl.
The term xe2x80x9cHetxe2x80x9d as used herein means a monovalent radical derived by removal of a hydrogen from a five- or six-membered, saturated or unsaturated heterocycle containing from one to three heteroatoms selected from nitrogen, oxygen and sulfur. Optionally, the heterocycle may bear one or two substituents; for example, N-oxido, lower alkyl, (C1-3)alkyl-phenyl, lower alkoxy, halo, amino or lower alkylamino. Again optionally, the five- or six-membered heterocycle can be fused to a second cycloalkyl, an aryl (e.g. phenyl) or another heterocycle.
Examples of suitable heterocycles and optionally substituted heterocycles include morpholine, thiadiazole, quinoline, 3,4-methylene-dioxyphenyl, benzothiazole, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, 1H-imidazole, 1-methyl-1H-imidazole, pyrazole, furan, thiophene, oxazole, isoxazole, thiazole, 2-methylthiazole, 2-aminothiazole, 2-(methylamino)-thiazole, piperidine, 1-methylpiperidine, 1-methylpiperazine, 1,4-dioxane, pyridine, pyridine N-oxide, pyrimidine, 2,4-dihydroxypyrimidine, 2,4-dimethylpyrimidine, 2,6-dimethylpyridine, 1-methyl-1H-tetrazole, 2-methyl-2H-tetrazole, benzoxazole and thiazolo[4,5-b]-pyridine.
The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d as used herein means a non-toxic, generally inert vehicle for the active ingredient which does not adversely affect the ingredient.
The term xe2x80x9ceffective amountxe2x80x9d means a predetermined antiviral amount of the antiviral agent, i.e. an amount of the agent sufficient to be effective against the virus in vivo.
The compounds of formula (I) can be obtained in the form of therapeutically acceptable salts. The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d as used herein includes those derived from pharmaceutically acceptable bases. Examples of suitable bases include choline, ethanolamine and ethylenediamine. Na+, K+, and Ca++ salts are also contemplated to be within the scope of the invention (also see Pharmaceutical salts, Birge, S. M. et al., J. Pharm. Sci. (1977), 66, 1-19, incorporated herein by reference).
The term xe2x80x9cpharmaceutically acceptable esterxe2x80x9d as used herein, either alone or in combination with another radical, means esters of the compound of formula (I) in which the carboxyl function is replaced by an alkoxycarbonyl function: 
in which the R moiety of the ester is selected from alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl); alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl (e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted with halogen, C1-4 alkyl or C1-4 alkoxy. Other suitable prodrug esters can be found in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985) incorporated herewith by reference. Such pharmaceutically acceptable esters are usually hydrolyzed in vivo when injected in a mammal and transformed into the acid form of the compound of formula (I).
With regard to the esters described above, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 16 carbon atoms, particularly 1 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
In particular the esters may be a C1-6 alkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted with at least one halogen, C1-6 alkyl, C1-6 alkoxy, nitro or trifluoromethyl.
Preferred Embodiments
According to a first embodiment of this invention, preferably compounds of the invention are those in which ring A is a benzene ring, as represented by the formula Ixe2x80x2: 
Wherein X, W, R1, Y and R3 are as defined above, with the provisos indicated above for formula (I). The compounds of formula Ixe2x80x2 exist in forms (1), (2) and (3), as described for the compounds of formula I.
Alternatively preferably, compounds of this invention are those in which ring A is a five-membered ring containing a sulfur atom, as represented by the formulae Ixe2x80x3and Ixe2x80x2xe2x80x3: 
Wherein X, W, R1, Y and R3 are as defined above, without the provisos indicated above for formula (I). The compounds of formulae Ixe2x80x3 and Ixe2x80x2xe2x80x3 exist in forms (1), (2) and (3), as described for the compounds of formula I.
Alternatively even more preferably, compounds of the invention have the following formula: 
wherein R3 and R5 are as defined above, without the provisos indicated above for formula (I).
The compounds of the present invention can be synthesized as racemic mixtures and then separated in their respective single diastereoisomers. All such diastereoisomers are contemplated within the scope of the present invention.
Preferably, such diastereoisomers include mixture of compounds with the following relative stereochemistry between [Y and C(O)NHxe2x80x94R3] and [C(O)NHxe2x80x94R3 and R4]: 
Formulas (Ia) and (Ib) both represent racemic mixtures of compounds with the relative stereochemistry referred to as xe2x80x9ccis/cisxe2x80x9d. 
Formulas (Ic) and (Id) both represent racemic mixtures of compounds with the relative stereochemistry referred to as xe2x80x9ctrans/transxe2x80x9d. 
Formulas (Ie) and (If) both represent racemic mixtures of compounds with the relative stereochemistry referred to as xe2x80x9ctrans/cisxe2x80x9d. 
Formula (Ig) and (Ih) both represent racemic mixtures of compounds with the relative stereochemistry referred to as xe2x80x9ccis/transxe2x80x9d.
More preferably, such diastereoisomers include mixture of compounds with the relative stereochemistry xe2x80x9ccis/cisxe2x80x9d: 
Also preferred are diastereoisomers with the relative stereochemistry xe2x80x9ctrans/transxe2x80x9d: 
Most preferably, compounds of formula (I), present in an xe2x80x9ccis/cisxe2x80x9d relative stereochemistry that can also be represented as follows: 
Still most preferably, the invention comprises pure enantiomers of compounds of formula (Ia) or (Ib) with the relative stereochemistry xe2x80x9ccis/cisxe2x80x9d: 
With respect to compounds of formulae I, Ixe2x80x2, Ixe2x80x3, Ixe2x80x2xe2x80x3, Ia, Ib, Ic, Id, Ie, If, Ig, and Ih, preferably X is H and W is OH; or X and W form a carbonyl group. Most preferably, X and W form a carbonyl group.
With respect to compounds of the formulae Ixe2x80x2, Ixe2x80x3 and Ixe2x80x2xe2x80x3 preferably A is phenyl or thiophene. Most preferably, A is thiophene.
With respect to compounds of the formulae Ixe2x80x2, Ixe2x80x3 and Ixe2x80x2xe2x80x3 preferably R1 is H; or one or two substituents independently selected from the group consisting of: hydroxy; halo; lower alkyl; lower alkoxy; lower thioalkyl; haloalkyl (e.g. trifluoromethyl); or xe2x80x94C(O)R2 wherein R2 is lower alkyl, aryloxy or benzyloxy.
More preferably, R1 is H, halo or C14 alkyl.
Even more preferably, R1 is H, fluoro or methyl.
Most preferably, R1 is H or methyl.
Preferably, Y is phenyl optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 is lower alkyl, lower cycloalkyl, lower alkoxy, halo, hydroxy, nitrile or trifluoromethyl, and R6 is lower alkyl, lower cycloalkyl, lower alkoxy, hydroxy or trifluoromethyl; said phenyl ring being optionally fused with a saturated or unsaturated 4 to 6-membered ring optionally containing a heteroatom selected from N, O and S; or Y is ethylene-phenyl, said ethylene moiety being optionally mono-substituted with lower alkyl, wherein said phenyl ring is optionally mono- or di-substituted with R5 or C(O)R6, wherein R5 and R6 are as defined above; said phenyl ring being optionally fused with a saturated or unsaturated 4- to 6-membered ring optionally containing a heteroatom selected from N, O and S.
More preferably, Y is naphthyl, CHxe2x95x90CH-phenyl, C(CH3)xe2x95x90CH-phenyl or phenyl, wherein the phenyl ring is optionally mono- or di-substituted at the 3, 4, or 5 position with R5, wherein R5 is halo, C1-4 alkyl, hydroxy, CF3 or NHC(O)-(lower alkyl).
Still more preferably, Y is phenyl optionally substituted with: 3,4-Cl; 3-F,4-Cl; 3-Cl,4-F; 3,4-Br; 3-F,4-CH3; 3,4-CH3; 3-CF3, NHC(O)xe2x80x94(CH2)3CH3 and 
Most preferably, Y is phenyl optionally substituted with:
3,4-Cl and 3,4-Br.
Preferably, R3 is selected from the group consisting of:
cyclohexyl; C1-6 alkyl; C1-6 thioalkyl; (C1-6 alkyl)phenyl wherein the phenyl ring is optionally substituted with:
lower alkyl, CF3, halo, CN, azido, lower alkoxy, (lower alkyl)acyl, C1-6 thioalkyl, C1-6 alkylsulfonyl, NHC(O)-lower alkyl, aryl, aryloxy, hydroxy, nitro, amino, or Het, said Het optionally mono- or di-substituted with lower alkyl, lower alkoxy, halo, hydroxy, nitrile, trifluoromethyl; 
Even more preferably, R3 is selected from the group consisting of: C1-6 alkyl; C1-6 thioalkyl; 
Most preferably, R3 is selected from the group consisting of: 
Particularly preferred compounds of the invention are compounds having the formula Ixe2x80x3. Of compounds having the formula Ixe2x80x3, those having the xe2x80x9ccis/cisxe2x80x9d configuration are particularly preferred.
Preferably, R4 is a carboxylic acid, a salt or an ester thereof.
Different Forms of Compounds of Formula (I)
Compounds of formula (I) according to the invention can present themselves in different forms according to the solvent and the pH in which they are dissolved. For example, compound 1001 (Table 1, form 1) can exist in equilibrium with compound 2001 (see Table 2, hereinafter) and compound 3005 (Table 3) in form (3) when dissolved in phosphate buffer at pH 7.4. Without wishing to be bound by theory, it is believed that the predominant form in solution at pH 7.4 is represented by form (1). 
Specific Embodiments
Included within the scope of this invention are all compounds of formula formulae I, Ixe2x80x2, Ixe2x80x3, Ixe2x80x2xe2x80x3, Ia, Ib, I,c, Id, Ie, If, Ig, or Ih, as presented in Tables 1 to 10 (with the exception of those compounds excluded by provisos).
Anti-papilloma Virus Activity
The antiviral activity of the compounds of formula (I) can be demonstrated by biochemical and biological procedures showing the inhibitory effect of the compounds on DNA replication.
Preferably, the compounds of formula (I) as described above are inhibitory against human papillomavirus (HPV). More preferably the compounds are active against HPV low risk or high risk type. Even more preferably, against low risk type HPV (i.e. type 6, type 11 and type 13, and especially HPV type 11). Alternatively, the high-risk type is selected from the group consisting of types 16, 18, 31, 33, 35, 45, 52, or 58, preferably, type 16). Most preferably, the compounds of the invention are directed against HPV types 6 and 11, even most preferably, against HPV-11.
A biochemical procedure for demonstrating anti-papilloma virus activity for the compounds of formula (I) is described in the examples hereinafter. This particular assay determines the ability of a test compound to inhibit the activity (IC50) of HPV-11 DNA replication. More specifically, in the assay described herein, the inhibitory activity of the test compound is evaluated based on its ability to interfere with the E1-E2-DNA origin of replication interaction, thereby inhibiting initiation of viral DNA replication.
Methods for demonstrating the inhibitory effect of the compounds of formula (I) on papilloma viral replication involving in vitro assays are described in Examples 11 to 15 herein.
When a compound of formula (I), or one of its therapeutically acceptable salts, is employed as an antiviral agent, it may be administered orally, topically or systemically to mammals, e.g. humans, rabbits or mice, alone or in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice.
Whether it be termed treatment or prophylaxis, a compound of formula (I) may also be used to prevent perinatal transmission of HPV from mother to baby, by administration to the mother prior to giving birth. More specifically, a compound of formula (I) may be used to prevent laryngeal papillomatosis in the baby.
For oral administration, the compound or a therapeutically acceptable salt thereof can be formulated in unit dosage forms such as capsules or tablets each containing a predetermined amount of the active ingredient, ranging from about 25 to 500 mg, in a pharmaceutically acceptable carrier.
For topical administration, the compound may be formulated in pharmaceutically accepted vehicles containing 0.1 to 5 percent, preferably 0.5 to 5 percent, of the active agent. Such formulations can be in the form of a solution, cream or lotion.
For parenteral administration, the compound of formula (I) may be administered by either intravenous, subcutaneous or intramuscular injection, in compositions with pharmaceutically acceptable vehicles or carriers. For administration by injection, it is preferred to use the compounds in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.
Suitable vehicles or carriers for the above noted formulations are described in standard pharmaceutical texts, e.g. in xe2x80x9cRemington""s The Science and Practice of Pharmacyxe2x80x9d, 19th ed., Mack Publishing Company, Easton, Pa., 1995, or in xe2x80x9cPharmaceutical Dosage Forms And Drugs Delivery Systemsxe2x80x9d, 6th ed., H. C. Ansel et al., Eds., Williams and Wilkins, Baltimore, Md., 1995.
The dosage of the compound will vary with the form of administration and the particular active agent chosen. Furthermore, it will vary with the particular host under treatment. Generally, treatment is initiated with small increments until the optimum effect under the circumstance is reached. In general, the compound of formula I is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.
For oral administration, the compound or a therapeutically acceptable salt may be administered in the range of 10 to 200 mg per kilogram of body weight per day, with a preferred range of 25 to 150 mg per kilogram.
For topical application, the compound of formula (I) may be administered in a suitable formulation to the infected area of the body e.g. the skin, the genitalia, in an amount sufficient to cover the infected area. The treatment may be repeated, for example, every four to six hours until lesions heal.
For systemic administration, the compound of formula (I) may be administered at a dosage of 10 mg to 150 mg per kilogram of body weight per day, although the aforementioned variations will occur. However, a dosage level that is in the range of from about 10 mg to 100 mg per kilogram of body weight per day is most desirably employed in order to achieve effective results.
Although the formulations disclosed herein are indicated to be effective and relatively safe medications for treating papilloma viral infections, the possible concurrent administration of these formulations with other medications or agents to obtain beneficial results is also contemplated. Such other medications or agents include TCA, podophyllin, podofilox, Interferon or Imiquimod.
In addition to the above-mentioned antiviral agents, the compounds according to the invention may also be used post-cryotherapy or post-surgery or in combination with any other treatment for physically removing warts.
Methodology and Synthesis
The synthesis of compounds of formula Ixe2x80x2 is illustrated in Scheme I. The radicals Y, R3 and R4 are as defined previously: 
A): Commercially available indan-1,3-dione (i) [or prepared according to known literature procedure: D. R. Bukle, N. J. Morgan, J. W. Ross, H. Smith, B. A. Spicer; J. Med. Chem. 1973, 16, 1334-1339] is condensed with aldehyde (ii) in a protic solvent (e.g. ethanol or propanol) in the presence of a catalytic amount of an organic amine (e.g. piperidine) to form the benzylidene (iii).
B): Benzylidene (iii) is converted to the epoxide (iv) by base-catalyzed oxidation with hydrogen peroxide in a protic solvent (such as methanol).
C): Epoxide (iv) undergoes thermal 1,3-dipolar cycloaddition in the presence of maleimide (v) at temperatures ranging from 80 to 100xc2x0 C. in a solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina, L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11, 1463-1466). Thus racemic xe2x80x9ccis/cisxe2x80x9d (vi) and racemic xe2x80x9ccis/transxe2x80x9d (vii) are obtained after purification (crystallization, flash column chromatography, or preparative HPLC). In general maleimides such as (v) are commercially available or alternatively can be easily prepared using literature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J. Org. Chem., 1997, 62, 2652-2654).
D): Racemic xe2x80x9ccis/cisxe2x80x9d compound (vi) is hydrolyzed to yield its opened carboxylate form (viii) also as xe2x80x9ccis/transxe2x80x9d racemic mixture. Hydrolysis is achieved under aqueous basic conditions, such as aqueous sodium hydroxide and acetonitrile as a co-solvent.
Alternatively steps A) and B) can be carried out as a xe2x80x9cone potxe2x80x9d reaction using an appropriate solvent (e.g. propanol) as described in Scheme II: 
A): Diazoindan-1,3-dione (ix) [prepared according to literature procedure: J. Chem. Soc. Chem. Commun., 1990, 652-653] was reacted with aldehyde (ii) and maleimide (v) in the presence of a catalytic amount of rhodium(II) to give racemic xe2x80x9ccis/cisxe2x80x9d compound (vi).
B): The corresponding carboxylate (viii) is made following the hydrolysis procedure described in Scheme I, step D).
C) This step presents a further method for synthesizing compounds of formula Ixe2x80x2 in an alternative closed form. Racemic xe2x80x9ccis/cisxe2x80x9d compound (vi) is first hydrolyzed using procedure described in Scheme I, step D, followed with treatment with acid using dilute aqueous HCl to produce hydroxylactone (x) as racemic xe2x80x9ccis/cisxe2x80x9d.
Cxe2x80x2) Alternatively the sodium salt intermediate (viii) is passed through a reverse phase column (HPLC) using a trifluoroacetic acid containing eluent to yield the hydroxylactone (x).
Compounds of formula Ixe2x80x2 wherein X and W form an epoxide are synthesized as illustrated in Scheme III: 
A): Racemic xe2x80x9ccis/cisxe2x80x9d compound (vi) is converted to the desired inhibitors via first hydrolysis under basic conditions, following by acidification and treatment with diazomethane. Compounds (xi), (xii) and (xiii) are separated from the mixture by flash chromatography or by preparative HPLC.
Scheme IV illustrates a general method for the synthesis of compounds of formula Ixe2x80x2 wherein X is H and W is hydroxy: 
A): Reduction of racemic xe2x80x9ccis/cisxe2x80x9d compound (vi) is achieved using a hydride source (e.g. sodium borohydride) to give mixtures of the monohydroxy derivatives (xiv and xv) in addition to the hydroxy lactone (xvi) having the relative stereochemistry as shown.
B): After separation, racemic (xiv and xv) are hydrolyzed using the same procedure as in Scheme I, step D) to give racemic (xvii and xviii) after preparative separation.
Scheme V illustrates the method for synthesizing compounds of formula Ixe2x80x2 with the relative stereochemistry in trans/trans. 
A): The amide (xix) obtained using literature procedure (ex: G. B. Villeneuve and T. H. Chan Tetrahedron Letters, 1997, 38,6484) is reacted with epoxide (iv) in toluene under refluxing conditions to yield the cycloadduct ester (xx) as racemic trans/trans isomers.
B): Hydrolysis of the ester (xx) is done as described in Scheme I, step D) to give the desired carboxylate (xxi) also as racemic trans/trans isomers.
Compounds of formula Ixe2x80x3 and of formula Ixe2x80x2xe2x80x3, may be made in an analogous manner to those of formula Ixe2x80x2, except that instead of indan-1,3-dione as starting material, compound xxii is used. 
A): Compound(xxii) [prepared by homologation of commercially available 5-methyl-2-thiophenecarboxaldehyde with malonic acid, followed by reduction of the exocyclic double bond with sodium amalgam, or hydrogen over palladium, followed by cyclization with oxalyl chloride/AlCl3, or polyphosphoric acid, followed by oxidation with CrO3/t-butylhydroperoxide] is condensed with aldehyde (ii) in a protic solvent (e.g. ethanol or propanol) in the presence of a catalytic amount of an organic amine (e.g. piperidine) to form the benzylidene (xxiii).
B): Benzylidene (xxiii) is converted to the epoxide (xxiv) by base-catalyzed oxidation with hydrogen peroxide in a protic solvent (such as methanol).
C): Epoxide (xxiv) undergoes thermal 1,3-dipolar cycloaddition in the presence of maleimide (v) at temperatures ranging from 80 to 100xc2x0 C. in a solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina, L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11, 1463-1466). Thus racemic xe2x80x9ccis/cisxe2x80x9d (xxvi) and racemic xe2x80x9ccis/transxe2x80x9d (xxvii) are obtained after purification (crystallization, flash column chromatography, or preparative HPLC). In general maleimides such as (v) are commercially available or alternatively can be easily prepared using literature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J. Org. Chem., 1997, 62, 2652-2654).
D): Racemic xe2x80x9ccis/cisxe2x80x9d compound (xxvi) is hydrolyzed to yield its opened carboxylate form (xxviii) also as xe2x80x9ccis/cisxe2x80x9d racemic mixture. Hydrolysis is achieved under aqueous basic conditions, such as aqueous sodium hydroxide and acetonitrile as a co-solvent.
An alternate route to compounds of the formula Ixe2x80x3 is shown in Scheme VII. 
A) Compound xxix [prepared by homologation of commercially available -5-methyl-2-thiophenecarboxaldehyde with malonic acid, followed by reduction of the exocyclic double bond with sodium amalgam, or hydrogen over palladium, followed by cyclization with oxalyl chloride/AlCl3, or polyphosphoric acid] is condensed with aldehyde (ii) in the presence of a catalytic amount of an acid catalyst (e.g. p-toluene sulfonic acid) in benzene or toluene, to form the benzylidene (xxx).
B) Benzylidene (xxx) is converted to the epoxide (xxxi) by oxidation (e.g. CrO3/t-butylhydroperoxide)
C) Epoxide (xxxi) undergoes thermal 1,3-dipolar cycloaddition in the presence of maleimide (v) at temperatures ranging from 80 to 100xc2x0 C. in a solvent such as toluene or xylene (ref.: M. Y. Krysin, I. K. Anohina, L. P. Zalukaev; Khimiya Geterotsiklicheskikh Soedinenii, 1987, 11, 1463-1466). Thus racemic xe2x80x9ccis/cisxe2x80x9d (xxxii) and racemic xe2x80x9ccis/transxe2x80x9d (xxxiii) are obtained after purification (crystallization, flash column chromatography, or preparative HPLC). In general maleimides such as (v) are commercially available or alternatively can be easily prepared using literature procedures (e.g. P. Y. Reddy, S. Kondo, T. Toru, Y. Ueno; J. Org. Chem., 1997, 62, 2652-2654).
D) Racemic xe2x80x9ccis/cisxe2x80x9d compound (xxxii) is hydrolyzed to yield its opened carboxylate form (xxxiv) also as xe2x80x9ccis/cisxe2x80x9d racemic mixture. Hydrolysis is achieved under aqueous basic conditions, such as aqueous sodium hydroxide and acetonitrile as a co-solvent.